A high performance liquid chromatography tandem mass spectrometric (HPLC-MS/MS) method was developed for the determination of nifursol metabolite 3,5-dinitrosalicylic acid hydrazide(DNSAH) residue in animal tissues.The sample was extracted with ethyl acetate after hydrolysised and derivatized,then cleaned up by n-hexane.The condensed sample solution was separated on a reversed-phase column of Thermo Hypersil Gold C18(100 mm×2.1 mm i.d.，1.9 μm)by gradient elution with methanol-water as mobile phase at a flow rate of 0.3 mL/min.The quantitation detection was performed by HPLC-tandem MS using multiple reaction monitoring(MRM) under negative ion electrospray ionization.The calibration curve of DNSAH was linear in the range of 0.5-5.0 μg/kg.The limit of quantitation was 0.5μg/kg.The recoveries at three spiked levels were between 91% and 103% with RSDs(n=6) of 3.7%-13.6%.The method was sensitive and accurate,and could meet the requirements of the domestic and international legislations.Therefore,the method was suitable for the determination and confirmation of residue of nifursol metabolite in animal tissues.